PerfeCTa SYBR® Green FastMix

Super. Fast. PerfeCTa.

Features & Benefits

  • 2X concentrated master mixes with stabilized SYBR Green dye and exceptional temperature stability (≥30 days at 22°C) that withstand repetitive freeze-thaw (≥ 20X).
  • Rigorously optimized for both fast and conventional thermal cycling parameters.
  • Supports efficient vortex mixing with proprietary anti-foaming technology.
  • Maximizes assay sensitivity and target precision with highly modified Taq DNA polymerase and stringent ultrapure, AccuFast™ II antibody hotstart technology

 

PerfeCTa SYBR Green FastMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Description

PerfeCTa SYBR Green FastMix is a 2X concentrated, ready-to-use reaction cocktail that contains all components, except primers and DNA template. This rigorously optimized master mix contains of proprietary buffer technology, stabilizers and AccuFast Taq DNA polymerase to deliver maximum assay precision, sensitivity, and PCR efficiency for accelerated or conventional thermal cycling conditions for SYBR Green detection. Dye-based detection methods are critically dependent on highly specific amplification because dsDNA dyes will bind to any amplicon, including off-target primer elongation and primer dimerization. AccuFast hot start Taq DNA polymerase contains a proprietary mixture of ultra-pure monoclonal antibodies that stringently suppress primer elongation prior to the initial PCR denaturation step and allows for setup and multi-day storage at ambient room temperature prior to thermal cycling. AccuFast provides rapid release of fully active enzyme to support accelerated thermal cycling conditions.

Performance Data

Comparison to Finnzymes, ADAR


PerfeCTa SYBR Green FastMix Comparison to DyNAmo Flash SYBR Green PCR Kit

RNA-specific adenosine deaminase (ADAR) was amplified from log-fold dilutions of total HeLa cell cDNA (100 ng to 10 pg) using PerfeCTa™ SYBR Green FastMix or the DyNAmo Flash SYBR Green PCR Kit (Finnzymes) according to each manufacturers protocol. Averaged plots for quadruplicate reactions for each input quantity are shown. Fusion of DNA-binding peptide to Tbr DNA pol results in lower specificity of the DyNAmo kit which is evident in false positive results for no template control (NTC) reactions. Chemically modified polymerase produces delayed Cts and lower signal strength compared to AccuStart™ Taq. Cycling conditions: Finnzymes: 95°C, 7 min followed by 40 cycles of 95°C, 10s; 60°C, 20s PerfeCTa™ SYBR Green FastMix: 95°C, 20s followed by 40 cycles of 95°C, 1s; 60°C, 20s


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Comparison to Takara, ADAR


PerfeCTa SYBR Green FastMix Comparison to SYBR PreMix Ex Taq

RNA-specific adenosine deaminase (ADAR) was amplified from log-fold dilutions of total HeLa cell cDNA (100 ng to 10 pg) using PerfeCTa&trade; SYBR Green FastMix or SYBR PreMix Ex Taq&trade; (Takara) according to each manufacturers protocol. Averaged plots for quadruplicate reactions for each input quantity are shown. PerfeCTa&trade; SYBR Green FastMix produces higher fluorescent signal and detection of equal target amounts at earlier Cts. Cycling conditions for both kits: 95&deg;C, 20s followed by 40 cycles of 95&deg;C, 1s; 60&deg;C, 20s


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Comparison to SYBR GreenER, ADAR


PerfeCTa SYBR Green FastMix comparison to SYBR GreenER qPCR SuperMix

RNA-specific adenosine deaminase (ADAR) was amplified from log-fold dilutions of total HeLa cell cDNA (100 ng to 1 pg) using PerfeCTa&trade; SYBR Green FastMix or the SYBR GreenER qPCR SuperMix (Invitrogen) according to each manufacturers protocol. Averaged plots for quadruplicate reactions for each input quantity are shown. Replicate CT values are shown on the standard curve (Panel A, inset). Cycling conditions: Invitrogenn: 95&deg;C, 10 min followed by 40 cycles of 95&deg;C, 10s; 60&deg;C, 60s; PerfeCTa&trade; SYBR Green FastMix: 95&deg;C, 20s followed by 40 cycles of 95&deg;C, 1s; 60&deg;C, 20s. PerfeCTa SYBR Green FastMix amplified the ADAR gene with higher efficiency and greater sensitivity. All replicate reactions for SYBR GreenER qPCR SuperMix failed to amplify ADAR from 1 pg of cDNA.


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Details

  • Contents

    Single-tube, 2X concentrated reagent containing:

    • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, and dTTP.
    • AccuStart II Taq DNA Polymerase
    • SYBR Green I dye
    • Inert AccuVue dye
    • Proprietary enzyme stabilizers and performance-enhancing additives.
    • Titrated reference dye (if applicable)
  • Storage & Handling
    PerfeCTa SYBR Green FastMix is stable for 1 year when stored in a constant temperature freezer at -20°C, protected from light. For convenience, it may be stored unfrozen at 4°C for up to 6 months. After thawing, mix thoroughly before using. Stabilized reagent demonstrates no functional loss of performance after 20 freeze-thaw cycles or 2 months at 20°C.
  • Instrument Capability
    ROX
    • Applied Biosystems 5700
    • Applied Biosystems 7000
    • Applied Biosystems 7300
    • Applied Biosystems 7700
    • Applied Biosystems 7900
    • Applied Biosystems 7900HT
    • Applied Biosystems 7900 HT Fast
    • Applied Biosystems StepOne™
    • Applied Biosystems StepOnePlus™
    Low ROX
    • Applied Biosystems 7500
    • Applied Biosystems 7500 Fast
    • Stratagene Mx3000P®
    • Stratagene Mx3005P™
    • Stratagene Mx4000™
    • Applied Biosystems ViiA 7
    • Applied Biosystems QuantStudio™
    • Agilent AriaMx
    • Douglas Scientific IntelliQube®
    • QIAGEN Rotor-Gene Q
    No ROX
    • BioRad CFX
    • Roche LightCycler 480
    • Other
    Bio-Rad iCycler iQ systems
    • BioRad iCycler iQ™
    • BioRad MyiQ™
    • BioRad iQ™5
  • Related Resources
    Product Manuals
    Product Flyers
    Safety Data Sheets (SDS)
    CofA (PSF)
    Publications
    Allelic variants of the aryl hydrocarbon receptor differentially influence UVB-mediated skin inflammatory responses in SKH1 mice
    Kayla J. Smith, Toxicology - 2017
    Abstract
    The mouse strain SKH1 is widely used in skin research due to its hairless phenotype and intact immune system. Due to the complex nature of aryl hydrocarbon receptor (AHR) function in the skin, the development of additional in vivo models is necessary to study its role in cutaneous homeostasis and pathology. Variants of the Ah allele, exist among different mouse strains. The Ahb−1 and Ahd alleles express high and low affinity ligand binding forms of the AHR, respectively. The outbred SKH1 mice express the Ahb−2 and/or Ahd alleles. SKH1 mice were crossed with C57BL/6J mice, which harbor the Ahb−1 allele, to create useful models for studying endogenous AHR function. SKH1 mice were bred to be homozygous for either the Ahb−1 or Ahd allele to establish strains for use in comparative studies of the effects of differential ligand-mediated activation through gene expression changes upon UVB exposure. Ahb−1 or Ahd allelic status was confirmed by DNA sequence analysis. We tested the hypothesis that SKH1-Ahb−1 mice would display enhanced inflammatory signaling upon UVB exposure compared to SKH1-Ahd mice. Differential basal AHR activation between the strains was determined by assessing Cyp1a1 expression levels in the small intestine, liver, and skin of the SKH1-Ahb−1 mice compared to SKH1-Ahd mice. To determine whether SKH1-Ahb−1 mice are more prone to a pro-inflammatory phenotype in response to UVB, gene expression of inflammatory mediators was analyzed. SKH1-Ahb−1 mice expressed enhanced gene expression of the chemotactic factors Cxcl5, Cxcl1, and Ccl20, as well as the inflammatory signaling factors S100a9 and Ptgs2, compared to SKH1-Ahd mice in skin. These data supports a role for AHR activation and enhanced inflammatory signaling in skin.
    Uptake and biological responses in land snail Cornu aspersum exposed to vaporized CdCl2
    L. Sturba, Ecotoxicology and Environmental Safety - 2017
    Abstract
    The uptake of Cd and some biomarkers of exposure and effects have been investigated in specimens of land snail Cornu aspersum exposed to vaporized CdCl2 (10mg/L) for 7 days. The Cd levels quantified in snail's whole bodies confirmed Cd bioavailability trough vaporization and an higher accumulation in the midgut gland compared to the foot. Biological responses investigated showed a reduction of destabilization time of lysosomal membranes (NRRT) in hemocytes and an induction of catalase activities (CAT) in midgut gland. A further evidence of CdCl2 vaporized exposure was given by an increase in MT protein content as well as induction of Cd-MT gene expression, highlighting the central role of the midgut gland in Cd detoxification. These biomarkers can thus be considered as sensitive tools for the assessment of Cd contamination in the air using land snails as bioindicators. No changes in of GST activity and MDA were observed. From the overall results, the land snail, C. aspersum, could be used as good bioindicator of air quality for pollution monitoring purposes having shown clear signs of exposure and effects due Cd exposure by air.
    Loop-mediated isothermal DNA amplification for asymptomatic malaria detection in challenging field settings: Technical performance and pilot implementation in the Peruvian Amazon
    Elisa Serra-Casas, PLOS ONE - 2017
    Abstract
    Background Loop-mediated isothermal DNA amplification (LAMP) methodology offers an opportunity for point-of-care (POC) molecular detection of asymptomatic malaria infections. However, there is still little evidence on the feasibility of implementing this technique for population screenings in isolated field settings. Methods Overall, we recruited 1167 individuals from terrestrial (‘road’) and hydric (‘riverine’) communities of the Peruvian Amazon for a cross-sectional survey to detect asymptomatic malaria infections. The technical performance of LAMP was evaluated in a subgroup of 503 samples, using real-time Polymerase Chain Reaction (qPCR) as reference standard. The operational feasibility of introducing LAMP testing in the mobile screening campaigns was assessed based on field-suitability parameters, along with a pilot POC-LAMP assay in a riverine community without laboratory infrastructure. Results LAMP had a sensitivity of 91.8% (87.7–94.9) and specificity of 91.9% (87.8–95.0), and the overall accuracy was significantly better among samples collected during road screenings than riverine communities (p≤0.004). LAMP-based diagnostic strategy was successfully implemented within the field-team logistics and the POC-LAMP pilot in the riverine community allowed for a reduction in the turnaround time for case management, from 12–24 hours to less than 5 hours. Specimens with haemolytic appearance were regularly observed in riverine screenings and could help explaining the hindered performance/interpretation of the LAMP reaction in these communities. Conclusions LAMP-based molecular malaria diagnosis can be deployed outside of reference laboratories, providing similar performance as qPCR. However, scale-up in remote field settings such as riverine communities needs to consider a number of logistical challenges (e.g. environmental conditions, labour-intensiveness in large population screenings) that can influence its optimal implementation.
    Solar thermotherapy reduces the titer of Candidatus Liberibacter asiaticus and enhances canopy growth by altering gene expression profiles in HLB-affected citrus plants
    Melissa M Doud, Horticulture Research - 2017
    Abstract
    Huanglongbing (HLB), a systemic and destructive disease of citrus, is associated with ‘Candidatus Liberibacter asiaticus’ (Las) in the United States. Our earlier work has shown that Las bacteria were significantly reduced or eliminated when potted HLB-affected citrus were continuously exposed to high temperatures of 40 to 42 °C for a minimum of 48 h. To determine the feasibility and effectiveness of solar thermotherapy in the field, portable plastic enclosures were placed over commercial and residential citrus, exposing trees to high temperatures through solarization. Within 3–6 weeks after treatment, most trees responded with vigorous new growth. Las titer in new growth was greatly reduced for 18–36 months after treatment. Unlike with potted trees, exposure to high heat did not eradicate the Las population under field conditions. This may be attributed to reduced temperatures at night in the field compared to continuous high temperature exposure that can be maintained in growth chambers, and the failure to achieve therapeutic temperatures in the root zone. Despite the presence of Las in heat-treated commercial citrus, many trees produced abundant flush and grew vigorously for 2 to 3 years after treatment. Transcriptome analysis comparing healthy trees to HLB-affected citrus both before and after heat treatment demonstrated that post-treatment transcriptional expression patterns more closely resembled the expression patterns of healthy controls for most differentially expressed genes and that genes involved with plant-bacterium interactions are upregulated after heat treatment. Overall, these results indicate that solar thermotherapy can be an effective component of an integrated control strategy for citrus HLB.
    Protective role of Indoleamine 2,3 dioxygenase in Respiratory Syncytial Virus associated immune response in airway epithelial cells
    Devi Rajan, Virology - 2017
    Abstract
    RSV is a major cause of severe lower respiratory infection in infants and young children. With no vaccine yet available, it is important to clarify mechanisms of disease pathogenesis. Since indoleamine-2,3-dioxygenase (IDO) is an immunomodulatory enzyme and is upregulated with RSV infection, we studied it in vivo during infection of BALB/c mice and in vitro in A549 cells. RSV infection upregulated IDO transcripts in vivo and in vitro. IDO siRNA decreased IDO transcripts ~2 fold compared to control siRNA after RSV infection but this decrease did not affect RSV replication. In the presence of IFN-γ, siRNA-induced a decrease in IDO expression that was associated with an increase in virus replication and increased levels of IL-6, IL-8, CXCL10 and CCL4. Thus, our results show IDO is upregulated with RSV infection and this upregulation likely participates with IFN-γ in inhibition of virus replication and suppression of some host cell responses to infection.
    Click here to see all Publications
    FAQs
    Can I use PerfeCTa® SYBR® Green FastMix® instead of SYBR Green SuperMix?
    You can use FastMix instead of SuperMix. We have observed similar results with both PCR mixes, however, slightly lower background was observed using SuperMix when using 10 ng of cDNA or more in the qPCR. The qScript microRNA Quantification system has been validated using SYBR Green SuperMix.
    Click here to see all FAQs

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